A Space‐Dependent ‘Enzyme‐Substrate’ Type Probe based on ‘Carboxylesterase‐Amide Group’ for Ultrafast Fluorescent Imaging Orthotopic Hepatocellular Carcinoma

Abstract Fast and selective fluorescence imaging for a biomarker to related‐disease diagnosis remains a significant challenge due to complex physical environment. Human carboxylesterase (CE) is expected to be a potential biomarker of hepatocellular carcinoma (HCC) to improve the accuracy of diagnosis. However, existing probes for CE has slow response rate and low selectivity. Herein, the amide group is selected as CE‐responsive sites based on the “substrate‐hydrolysis enzymatic reaction” approach. From a series of off–on probes with leave groups in the amide unit, probe JFast is screened with the optimal combination of rapid response rate and high selectivity toward CE. JFast requires only 150 s to reach the maximum fluorescence at 676 nm in the presence of CE and free from the interference of other esterase. Computational docking simulations indicate the shortest distance between the CE and active site of JFast . Cell and in vivo imaging present that the probe can turn on the liver cancer cells and tumor region precisely. Importantly, JFast is allowed to specifically image orthotopic liver tumor rather than metastatic tumor and distinguish human primary liver cancer tissue from adjacent ones. This study provides a new tool for CE detection and promotes advancements in accurate HCC diagnosis.

Instruments. 1 H NMR and 13 C NMR spectra were recorded on the Bruker Avance-600 MHz and 151 MHz NMR spectrometers, respectively. ESI mass spectrometry was carried out on the AB Triple TOF 5600plus System. UV-visible spectra and steady-state emission experiments were performed on a Hitachi U-3900 spectrometer and a Hitachi F-7000 spectrometer, respectively. The cytotoxicity assay was measured by a Bio Tek ELX808 fully automated microplate reader. The cell imaging experiments were measured by Zeiss LSM880 Airyscan confocal laser scanning microscope. The in vivo imaging assays were performed in the Perkin Elmer Lumina LT small animal optical imaging system. The high-performance liquid chromatography (HPLC) experiments were performed on Shimadzu LC-20AT HPLC spectrometer.

Molecular docking simulations. Molecular docking was performed in this study with CDOCKER
in Discovery Studio 2020 software. Before docking, the ligands were converted to 3D structure in SDF format, and prepared using the Prepare Ligands protocol to remove duplicates and generate dominant conformations. The crystal structure of human CES1 (PDB ID: 5A7H) was also prepared using the Prepare Protein with expurgations of waters and the original ligands. All the parameters in this program were set to the default values. The active cavity of human CES1 was defined as the binding site, with all other parameters as default. As the probe J7 could not induce any response towards CES1, we concluded that the conformation of J7 is quite anastomotic with the active cavity. Hence, other probes similar to conformation of J7 were picked out. Based on these results, the -CDOCKER interaction values were obtained and the distances form the probe to Ser221 and His468 were measured.
Determination of the detection of limit. The detection limit was calculated based on the fluorescence titration of J Fast in the presence of CE. The fluorescence intensity of probe was measured and standard deviation of the blank measurement was achieved. The detection limit was calculated by using detection limit with the following equation: Detection limit = 3σ/k Where σ is the standard deviation of the blank measurement, k is the slope between the fluorescence intensity versus various CE concentrations.
Photostability test. J Fast , J Fast +CE，BB3 and ICG were dissolved in 1 mL PBS solution. Sample S4 tubes were exposed to 640 nm laser lamp (58 mW/cm 2 ) and kept some distance away from the lamp. A cold trap equipped with aqueous solution was placed between lamp and sample tubes. Absorbance spectra were measured of samples at intervals (5 min) for 1 h. HPLC analysis. J Fast , J6, J8, BB3, CE, J Fast +CE, J6+CE and J8+CE (CE final concentration of 10 U/mL, other substances final concentration of 20 μM, all injection volumes of 10 μL, 1% DMSO aqueous solution, v/v) were characterized by Shimadzu LC-20AT HPLC spectrometer, respectively. It should be noted that the J Fast +CE, J6+CE and J8+CE groups need to be injected after 5 min of mixing to give sufficient enzyme reaction time.
Cell culture. Human liver carcinoma HepG2 cells, human mammary adenocarcinoma MCF-7 cell line, human normal liver HL7702 cell lines, human colorectal carcinoma SW480 cells, mouse embryonic fibroblast NIH/3T3 cells and mouse hepatocarcinoma Hepa1-6 cells were cultured and maintained in DMEM medium (supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin) at 37 °C and 5% CO 2 in a humidified incubator. Human colorectal carcinoma HCT116 cells and HCT116(p53 -/-) cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. HCT116(p53 -/-) cells were the gifted from Dr. XingKang Wu from Shanxi University S1 . Other cell lines were provided by the Institute of Biochemistry and Cell Biology, SIBS, CAS (China).
Preparation of cell lysate and activity assay of CE in cell lysate. Strict operating procedures are performed according to the kit instructions.
Cytotoxicity assay. Cells Counting Kit-8 (CCK-8) assay was carried out to assess cytotoxicity of J Fast against HepG2 and Hepa1-6 cells. Cells (10 4 cells/well) were plated into 96-well plates and then allowed to adhere for 24 h. Subsequently, the cells were incubated with 0, 5, 10, 15 and 20 µM of J Fast at 37℃ in an atmosphere of 5% CO 2 for 24 h. The CCK-8 solution (10 µL) was added to each well and incubated for 1 h, then the optical density (OD) was measured at 450 nm using a microplate reader.
When the amount of the probe added was 0 µM, the cell viability value was set to 100%. The cell viability was calculated by use of equations shown below. Cell viability (%) = (Mean OD of sample × 100) / (Mean OD of the control group).
Fluorescence imaging of CE activity in a HepG2 subcutaneous tumor-bearing mouse model. All the animal experiments were performed by following the protocols approved by the S5 Beijing Viton River Experimental Technology Co., Ltd. The mice were housed under a 12-h light/dark cycle and were allowed free access to food and water. HepG2 cells (1.5 × 10 7 cells) were selected for transplantation into the axillae of mice. After fifteen days, tumor xenograft mice were given J Fast (200 μM, 20 μL) by intratumoral injection during mouse anesthesia. Images were taken by a small animal live imaging system at different time points (0, 1, 3, 6, 15, 27, 33, and 45 min) upon excitation at 640 nm. The above experiments were performed on two other subcutaneous tumor-bearing mice after replacing J Fast with J6 and J8, respectively.
Fluorescence imaging in the orthotopic liver tumor model. Nude BABL/c mice were anesthetized and opened via midline incision to expose the liver. Hepa1-6 cell suspension (1.5 × 10 7 cells) was slowly injected the left liver lobe of the mice, and the treated live was send back to the abdominal cavity. After 10 days of tumor formation in culture, the mice were dissected and the major organs (heart, liver, spleen, lung, and kidney) and tumor parts were removed, and the removed organs were sprayed with 200 μM of J Fast solution and then fluorescent imaging using a small animal live imaging system.
Tissue slice imaging of human hepatocellular carcinoma. All tissue slice samples from 6 patients (adjacent tissues, n = 3; tumor tissues, n = 3) were harvested from Shanghai outdo biotech co.,ltd. Data and samples were prospectively collected with local ethics committee approval at each center, and all patients gave their informed consent to participate in the research. Adjacent tissues were identified for longitudinal assessment of liver function. The imaging of CE in the tissue slice samples was carried out using the designed J Fast probe, and imaged in CLSM. Scheme S1. Synthetic route of J1-J12 and J Fast .

2． Summary of fluorescent probes for detection of CE
Synthesis details of J1. Ⅰ Basic Blue 3 (5 mmol, 1795 mg) and sodium carbonate (20 mmol, 2120 mg) were dissolved in 30 mL DCM. The mixture was stirred at 45℃ under the protection with nitrogen.
After 5 min, sodium hyposulfite (20 mmol, 3482 mg) dissolved in 20 mL water was slowly added, and then stirred for additional 30 min. The organic phase was isolated in an anaerobic environment and immediately proceeded to the next reaction step.

7． Additional Fluorescence Imaging in Living Cells
We detected the CE activity in co-culture model (HepG2 and HL7702 cells), as shown in Figure S43.
Because the morphological differences between the two types of cells were not obvious, HL7702 cells were specifically labelled with 5 µM NP1 dyes for 30 min (NP1, as a specific probe for H 2 O 2 , was reported in our previous work; Anal. Chem., 2014, 86, 9970-9976), before co-incubation with HepG2 cells. Then the adherent cells were incubated with 5 µM J Fast for 30 min. In the blue channel (445 ± 20 nm, λ ex = 405 nm), HL7702 cells can be verified (shown in yellow box, Figure S43), due to NP1 S33 staining. While a bright red fluorescence signal was monitored in HepG2 cells in white box. These data indicated that the probe may serve as a tool to distinguish normal cells (HL7702 cells) and cancer cells (HepG2 cells).

HepG2
Red Chanel Blue Chanel Brightfield Merge Figure S43. CLSM images of co-culture HepG2 and HL7702 cells incubated with J Fast . Before